Animal feed compositions containing phytase derived from transgenic alfalfa and methods of use thereof

ABSTRACT

A value-added composition of matter containing plant matter from transgenic alfalfa which expresses exogenous phytase activity is disclosed. The phytase activity is a gene product of an exogenous gene encoding for phytase which has been stably incorporated into the genome of alfalfa plants. The transgenic alfalfa expresses phytase activity in nutritionally-significant amounts, thereby enabling its use in animal feeds to eliminate the need for phosphorous supplementation of livestock, poultry, and fish feed rations.

This invention was made with United States government support awarded bythe following agencies:

DOE Grant #DE-FGO5-920R22072 (CPBR #OR22072-34);

DOE Grant #OR22072-14 (MTU #: P0016167);

USDA Grant #92-34190-6941 (Purdue AGR 593-0130-20; 593-0220-09);

USDA AGRICARS Grant #58-3655-6-132 (Koegel);

USDA AGRICCREE Grant #94-33120-0433, Hatch Grant #3194, N194; and

USDA AGRICARS Grant #58-3655-2-205, 58-3655-7-208 (Straub).

The United States has certain rights in this invention.

This is a Continuation-In-Part of pending application Ser. No.08/638,448, filed Apr. 26, 1996.

FIELD OF THE INVENTION

The invention is drawn to value-added animal feed compositions andadditives containing unprocessed or minimally processed matter fromtransgenic alfalfa which expresses exogenous phytase activity inconcentrations nutritionally significant in monogastric animals. Theinvention is further drawn to novel uses of the animal feedcompositions.

BIBLIOGRAPHY

Complete bibliographic citations of the references described herein canbe found in the Bibliography section, immediately preceding the claims.

DESCRIPTION OF THE PRIOR ART

Livestock production, especially large-scale commercial livestockproduction for human consumption, requires the use of vast amounts ofnutritionally balanced animal feed. Because of the large amounts of feedrequired to sustain commercial livestock production, world-wide researchefforts have been made to develop feedstock additives which maximize thebioavailability of nutritionally important elements and compounds foundin common animal feedstocks.

In the early 1950's, for instance, it was speculated that the dietaryrequirements of egg-laying fowl might be met by utilizing protein-richextracts from green plants. Hughes and Eyles (1953) describe a feedingtrial with laying hens which used dietary protein extracted from theleaves of green plants. The authors hypothesized that dietary proteincould be extracted from green plants in an economical fashion, therebyeasing the shortage and lowering the cost of high protein feed in GreatBritain.

In more recent years, with the development of sophisticated methods ofgenetic manipulation, transgenic plants which express nutritionallyimportant compounds have been developed. However, in order toeffectively utilize transgenic plants which express exogenous proteins,the transgenic plants must be more economical to use than the feedstocksor feedstock additives they are designed to replace.

Therefore, it is necessary to maximize the expression of the exogenousprotein while simultaneously stabilizing the beneficial activity of theprotein. Additionally, the exogenous expressed protein ideally should beutilizable with very little or no post-harvest processing of thetransgenic plant material. If the exogenous protein is expressed in onlysmall quantities, or if the transgenic plant material must beextensively processed prior to use, or if the exogenous protein lackssufficient stability in the harvested plant material, the slim profitmargins encountered in commercial feed production will dictate againstswitching to the use of transgenic plant material. In short, becausealternative sources of nutrients continue to be relatively cheap andwidely available, the positive economics of producing nutritionallyimportant feed additives in transgenic plants remains marginal unlessthe above criteria are present.

The remarkable progress in recombinant plant genetics has greatlyspurred new investigations into the economics of manufacturing,isolating, and using exogenous proteins expressed in transgenic herbageplants such as alfalfa. In effect, valuable recombinant proteinproducts, which are now produced by fermentation using transgenicmicroorganisms, might be economically produced using transgenic plantsrather than native or recombinant microbes.

Austin et. al. (1994) studied the production of industrial enzymes intransgenic alfalfa, a report of which appeared in the Annals of the NewYork Academy of Sciences. These investigators researched the feasibilityof producing industrially important enzymes using alfalfa plants as"factories." The focus of this study was whether, using geneticengineering technology, cloned genes for valuable enzymes could beexpressed and economically harvested from plant hosts. The concept iseconomically attractive because, assuming the heterologous gene can bestably incorporated, many herbage plants are perennial, hardy crops,which are capable of more than one harvest per year. In the case ofalfalfa specifically, in temperate climates such as those found in themidwestern United States, alfalfa does not require irrigation and iscapable of three or more harvests a year. Moreover, since alfalfa isleguminous, it grows well without nitrogen fertilizer.

The Austin et al. paper noted above used a reporter gene,β-glucuronidase (GUS), as a model system. The analysis concluded (asnoted generally above) that the concentration of the desired value-addedproduct (in this case, GUS) in the transgenic plant is most criticalvariable for economic viability. Analogous field studies for transgenicalfalfa which expresses α-amylase and manganese-dependent ligninperoxidase have also been reported by Austin et al. (1995).

An enzyme group of particular interest is the phytases. Phytases, moreproperly referred to as myo-inositol hexaphosphate phosphohydrolases,are a family of enzymes which catalyze the step-wise removal ofinorganic orthophosphate from phytic acid (myo-inositol1,2,3,4,5,6-hexakisphosphate). The economic interest in phytase is dueto its ability to increase the bio-availability of inorganic phosphorousin phytate-containing non-ruminant animal feeds. Currently, feed fornon-ruminant animals must be supplemented with inorganic phosphorousbecause these animal cannot utilize the phosphorous present as phytate.

While phytase occurs widely in both plants and microorganisms, theenzyme has been extensively studied mostly from the filamentous fungi,particularly the Aspergilli, notably A. ficuum, and A. nidulans. For anexcellent review of phytases and their action on phytic acid see Gibson,D. M. and Ullah, A. B. J. (1990), incorporated herein by reference.

Regarding the nucleotide sequences which encode phytase, several suchsequences have been identified, sequenced, and cloned into variousheterologous hosts. For instance, Van Gorcom et al., U.S. Pat. No.5,436,156, incorporated herein by reference in its entirety, describethe isolation and cloning from A. ficuum of a DNA sequence coding forphytase. The isolated nucleotide sequence was successfully cloned andinserted into a vector capable of transforming a microbial expressionhost. Specifically, the nucleotide sequence was first cloned using thebacteriophage lambda AFD01, and further sub-cloned into pAN 8-1 andpUC19. The construct was then used to transform various types offilamentous fungi. (See also, EP 0 420 358 A1, to the same inventor.)

Ehrlich et al. (1993), describe the cloning and sequencing of a gene fora second type of phytase, designated PhyB. This phytase was isolatedfrom A. niger NRRL 3135, and had a pH optimum of 2.5. PhyB was found tohave different properties from the previously known phytase PhyA, whichhas a pH optimum of 5.0.

European Patent Application EP 0 449 375 A2 (Pen et al.) describes theexpression of phytase in tobacco seeds and rapeseeds.

Likewise, Verwoerd et al. (1995) describe the production andaccumulation of phytase in the leaves of tobacco plants transformed witha phytase-coding gene of A. niger. This paper describes the constitutiveexpression of a phytase cDNA from transgenic tobacco plants. Theexogenous phytase enzyme was secreted into the extracellular fluid atconcentrations approximately 90 times higher than that in the total leafextract. The phytase produced by the transgenic tobacco plants wascompared to native Aspergillus phytase and found to have identicalactivities. During plant maturation, it was found that the phytaseproduced in the tobacco remained biologically active and accumulated inamounts up to 14.4% of the total soluble protein found in the tobacco.

As noted briefly, above, the economic interest in phytase is due to itsability to increase the bio-availability of inorganic phosphorous inphytate-containing animal feeds. The increase in intensive, large-scalelivestock production has resulted in increased environmental problems,specifically eutrophication, due to the tremendous amount of manureproduced in such enterprises. Phosphorous, an essential nutrient forboth ruminants and non-ruminants, is necessarily added to the basal feedprovided to livestock. Much of this feed material also contains largeamounts of phytate. Phytate acts as the primary storage form ofphosphorous in most green plant materials and can account for more than50% of the total phosphorous content of the plant material. However, inmonogastric animals, the phosphorous contained in phytate is poorlydigested and largely excreted. Consequently, animal feeds are regularlysupplemented with more easily assimilated forms of inorganic phosphorous(e.g., dicalcium phosphate). The excreted phytate, which contains largeamounts of phosphorous, increases phosphorous loading to theenvironment, with concomitant environmental degradation.

Additionally, phytate is generally considered an anti-nutritional factordue to its ability to chelate multivalent cations. For instance, phytatewill bind multivalent cations such as calcium, iron, manganese, andzinc, to form insoluble complexes. This reduces the bio-availability ofthese minerals, which are essential for proper growth and maturation.Further still, phytate also complexes with several different types ofproteins, thereby obstructing enzymatic protein digestion.

As a consequence, several prior art references describe the use ofphytase-containing compositions to increase the bio-availability of thephosphate contained in phytate.

For instance, European Patent Application EP 0 619 369 A1 (Vanderbeke etal.) describes a phytase-containing enzyme composition which remainsenzymatically active at the low pH's found in the digestive tract ofmonogastric animals. This composition contains a combination of phytasesand fungal acid phosphatases. The combination results in a synergisticeffect which enables the mixture to enzymatically degrade phytate atrelatively low pH. (See also, U.S. Pat. No. 5,443,979, to the sameinventors.)

In a study reported in the Proceedings of the 1995 Cornell NutritionConference for Feed Manufacturers, in Rochester, New York, Han et al.reported that corn-soybean meal diets supplemented with microbialphytase significantly improved the utilization of phosphorous from suchfeeds and also decreased the amount of phosphorous excreted from swineby up to 50%. These researchers concluded that the need for inorganicphosphorous supplementation in swine feed stocks can be partially ortotally eliminated by the addition of dietary microbial phytase duringthe growth and finishing phase of the swine. It should also be notedhowever, that the long term effects of phytase supplementation, if any,remain unknown.

Therefore, there remains a need for an affordable, renewable animal feedsupplement which enables livestock to more efficiently utilize inorganicphosphorous.

SUMMARY OF THE INVENTION

A first embodiment of the invention is an animal or fish feedcomposition comprising juice, concentrated juice, or dried juice oftransgenic alfalfa plants which constituitively express exogenousphytase activity at a concentration which is nutritionally significantin animals and fish in general, and monogastric animals in particular.

A second embodiment of the invention is an animal or fish feedcomposition comprising leafy plant material from transgenic alfalfaplants which constituitively express exogenous phytase activity at aconcentration which is nutritionally significant in animals,particularly monogastric animals.

A third embodiment of the invention is a feed composition which includeswhole non-transgenic alfalfa juice or leafy material from non-transgenicalfalfa to supply xanthophylls, dietary protein and other nutrients tothe ration, in combination with whole alfalfa juice, concentratedalfalfa juice, partially fractionated alfalfa juice, or leafy plantmaterial from transgenic alfalfa which expresses phytase activity tosupply phytase to the ration. The soluble portion of partiallyfractionated juice from the transformed plants contains essentially allof the phytase produced in the transformants.

Another embodiment of the invention is drawn to a feed composition fornon-fowl, monogastric livestock (swine in particular) which containspartially fractionated juice (as a fresh liquid, frozen, concentrated,or dried) from transgenic alfalfa which expresses phytase activity.Here, the partial fractionation serves to remove xanthopylls from thejuice. This is the preferred form of feed for non-fowl livestock becausexanthophyll supplementation is not necessary.

A still further embodiment of the invention is a method of feedinglivestock, poultry, or fish, in which one of the animal feeds describedherein which contains leafy plant material or juice from transgenicalfalfa is fed to livestock, poultry, or fish whereby the need tosupplement the feed with inorganic phosphorous is reduced or eliminatedentirely.

The value-added feed composition of the present invention isparticularly advantageous when added to non-ruminant animal feed. Theinvention allows non-ruminant animals to utilize phosphorous in the feedmore efficiently, thus greatly reducing or eliminating the need forphosphorous supplementation. In addition, use of the feed compositionreduces phosphorous loading to the environment, thereby reducingenvironmental contamination.

Another notable advantage of the disclosed composition is its use inpoultry feed. Because the composition contains high levels of pigmentssuch as xanthophylls, and high levels of phytase, poultry feed which issupplemented with the feed composition need not be supplemented withphosphorous or pigmenting agents.

Another distinct advantage of the composition is that it is avalue-added product as compared to the market value of the herbagematter of non-transformed alfalfa. This allows farmers and feedformulators to not only diversify their market offerings, but also toincrease gross profit margins on otherwise fungible commodity goods.

The principal aim of the invention is to provide a value-addedmulti-component protein concentrate feed additive from transgenicalfalfa which expresses exogenous phytase. The multi-componentcomposition contains phytase produced by the transgenic alfalfa,pigments such as xanthophylls, and dietary protein.

A still further aim of the present invention is to provide a supplementfor monogastric feed rations which can be prepared directly from alfalfaplant matter and which contains high levels of phytase, xanthophylls,and dietary protein.

Further aims, objects, and advantages of the above-describedmulti-component value-added composition will become apparent upon acomplete reading of the Detailed Description, drawings, and attachedclaims, below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flow chart describing the average yield of variousproducts isolated from transgenic alfalfa which expresses phytase. Theyields are listed on a per 1000 kilogram (kg) dry matter basis and, inparentheses, on a per acre expected annual yield basis.

FIG. 2 is a graph depicting the temperature stability of phytaseactivity from the transgenic alfalfa described herein after treatment at0° C. (empty squares), 30° C. (diamonds), and 50° C. (solid squares).

FIG. 3 is a graph depicting the phytase activity in basal poultry dietssupplemented with frozen alfalfa juice from transgenic alfalfa ("juice")and concentrated juice from transgenic alfalfa ("lyoph. juice")expressing phytase after storage for 1, 2, and 3 weeks at -20° C., 4°C., and 22° C. (10/15, 10/22, and 10/30 correspond to week 1, week 2,and week 3 measurements, respectively.)

FIGS. 4A, 4B, and 4C are schematic diagrams of the binary vector tDNA ofthe phytase expression constructs used to transform alfalfa as describedherein below. The promoters and structural genes are depicted as arrowswhich indicate the direction of transcription. Terminaters are depictedas boxes. NOS=nopaline synthase; NPT II=neomycin phosphotransferase;SSU=Arabidopsis thaliana Rubisco small subunit promoter; TML=tumormorphology left; 35S=cauliflower mosaic virus 35S promoter.

DETAILED DESCRIPTION OF THE INVENTION

Transgenic Alfalfa Expressing Phytase

An overriding motivation behind this invention is the use of commonagricultural plants which have been genetically engineered to functionas "enzyme factories." The use of common, albeit genetically engineered,plants in this fashion significantly impacts the economics ofagricultural production. In the case of transgenic alfalfa (thepreferred plant host) which expresses exogenous phytase, the inventionallows farmers to more effectively feed their non-ruminant livestock atreduced prices, and also reduces phosphorous loading to the environment.Additionally, the plant matter containing the phytase, as well asvalue-added concentrates and isolates manufactured from the plantmaterial, can be sold at a greater profit than non-transformed herbagematter.

In the present invention, transgenic alfalfa which expressesnutritionally-significant amounts of phytase activity is the preferredplant material. However, any suitably transformed plant species whichexpresses sufficient levels of phytase and which will not adverselyaffect livestock by any other mechanism will function with comparablesuccess. Alfalfa is the preferred plant host because alfalfa is a hardy,perennial plant, which grows well with minimal fertilization andirrigation. Alfalfa is also a very prolific plant. In temperate areassuch as those found in the midwestern United States, alfalfa will yieldthree or more harvests per growing season.

Preferably, the alfalfa is stably transformed to constituitively expresshigh levels of enzymatically active phytase. Phytase enzymes areproduced in relatively large quantities by many microorganisms, mostnotably filamentous fungi of the genus Aspergillus. Phytase is alsoproduced by bacteria such as Bacillus subtilis, yeasts such asSaccharomyces cerevisiae, and various strains of Pseudomonas. Among thefilamentous fungi, Aspergillus ficuum (A. ficuum) produces a phytasewhich has a particularly high specific activity and thermostability.Phytase of high specific activity is also produced by A. nidulans and A.niger. In the present invention, it is preferred that the alfalfa planthost be transformed with one or more genetic elements which encodefunctional phytase, and that the genetic elements originate from afilamentous fungi selected from the group consisting of A. ficuum, A.nidulans, and A. niger.

The translocated genes may also include upstream or downstreamregulatory elements, such as promoters for constituitive, growthstage-specific, or organ-specific expression, targeting sequences,secretory sequences, terminator sequences, polyadenylation signals, andthe like, which might function to control gene expression in theheterologous host plant. Such regulatory elements may be homologous(native) to the host plant or they may be heterologous. Several of thesetypes of regulatory sequences are known to those skilled in the art ofrecombinant plant genetics.

An illustrative method for producing transgenic plants, commonlyreferred to as the binary vector method, utilizes a strain ofAgrobacterium containing a vir plasmid (which includes genes forvirulence) and a second, compatible plasmid which contains the geneconstruct to be translocated.

Here, a double-stranded cDNA encoding phytase is prepared from MRNAisolated from a filamentous fungi of the genus Aspergillus, preferablyA. niger. For expression in seeds, the DNA construct can be linked withregulatory sequences from the gene encoding the 12S storage proteincruciferin from Brassica napus. The construct is then sub-cloned into abinary vector such as pMOG23 (E. coli. K-12 strain DH5α, Centraal Bureauvoor Schimmelcultures, Baarn, The Netherlands, Accession No. CBS102.90). The vector is then introduced into Agrobacterium tumefacienscontaining a disarmed Ti plasmid. This can be done by any number of wellknown means, such as by electroporation. Bacterial cells containing theconstruct are co-cultivated with tissues from alfalfa or other plant tobe transformed, and transformed plant cells are selected using nutrientmedia containing antibiotics. The transformed cells are induced todifferentiate into plants on suitable nutrient media. The plants soproduced will produce seeds which contain and express the geneconstruct.

To produce plants which constituitively express the phytase geneconstruct, the phytase-encoding gene construct is preferably placedunder the regulatory control of the 35S promotor sequence of thecauliflower mosaic virus (CaMV). The construct is then sub-cloned into abinary vector as described above. The vector is then introduced intoAgrobacterium tumefaciens containing a disarmed Ti plasmid. Bacterialcells containing the construct are co-cultivated with tissues from thealfalfa to be transformed, and transformed plant cells are selectedusing nutrient media containing antibiotics. The transformed cells areinduced to differentiate into plants on suitable nutrient media. Theplants so produced constituitively express enzymatically active phytase.

The above method is illustrative only, and several other geneticengineering techniques which result in stable transgenic plants willfunction with equal success in the present invention. As noted above,several such methods are well known in the genetic engineering art. Themethod by which the transgenic plant starting material is constructed isnot critical for the performance of the present invention insofar as theresultant transgenic alfalfa expresses a relatively large amount offunctional exogenous gene product.

For instance, in the present invention, the preferred transgenic alfalfawas produced as follows: Binary vectors carrying phytase expressioncassettes were transformed into Agrobacterium tumefaciens strainLBA4404, facilitating Agrobacterium-meditated transformation of planttissue. The three constructs used are depicted in FIGS. 4A, 4B, and 4C.The constructs shown in FIGS. 4A and 4B contain the A. niger phytasegene placed under the control of the CaMV 35S promoter (FIG. 4A) or theArabidopsis thaliana Rubisco small subunit (SSU) promoter (FIG. 4B). Theconstructs of FIGS. 4A and 4B incorporate a signal peptide for targetingthe phytase enzyme to the apoplast. The expression cassettes were clonedinto derivative of the pBI binary vectors and mobilized intoAgrobacterium.

A third construct, depicted schematically in FIG. 4C, was fabricated inwhich the hybrid "MAC" promoter was used (pTZ117). This promotercontains distal elements of the CaMV 35S promoter, including thetranscriptional enhancer (-940 to -90, relative to the mRNA start site).The proximal promoter elements of MAC are derived from the Agrobacteriummannopine synthase promoter (-301 to +65 relative to the mRNA startsite). MAC has been reported to result in higher levels of expressionthan either of the natural promoters. (See Comai et al. (1990).) The MACpromoter was fused to the A. niger phytase gene, along with a signalpeptide for apoplast localization as in the first two contructs. Thisexpression cassette was cloned in a pCGN binary vector and mobilizedinto Agrobacterium.

In vitro transformation of both tobacco and alfalfa was accomplishedwith all three constructs. Significant levels of phytase expression wereobtained in all cases. The phytase expressed appeared underglycosylatedin both alfalfa and tobacco, but the enzyme retained stability at hightemperature (55° C.) and low pH (pH 2.5).

In transgenic alfalfa, the transformants containing the CaMV 35Spromoter gave the best levels of phytase production. Phytase expressionin these transformants yielded phytase concentrations ranging frombetween about 0.1% to about 2.0% of total soluble protein found in thetransformed plants. Extensive testing of transformed alfalfa usingstandardized assays for phytase activity indicated that thetransformants expressed phytase constituitively; in all transformants,increased phytase activity was detected throughout the entire plant.

Juicing the Alfalfa

In order to reduce the amount of material to be handled, phytase presentin the transgenic alfalfa may be concentrated by wet fractionation ofthe freshly harvested alfalfa. Here, the transgenic plant material isharvested, macerated, and fractionated by known means. If furtherclarification is desired, the juice may be heated by passing alternatingcurrent (AC) electricity through the juice or by other means of uniformheat addition. This causes aggregation of insoluble protein particleswithin the juice, which are then removed from the remaining solubleproteins (which solubles include the exogenous phytase activity). Ifdesired, the phytase within the soluble fraction may be furtherconcentrated by suitable means, such as ultrafiltration, dialysis, andthe like.

If the juice of the transgenic alfalfa is to be utilized, the followingprotocol is preferred. The juice is preferably made from alfalfa in thelate bud or early bloom stage. Juicing the alfalfa preferably occurs assoon as practicable after harvesting. It is preferred that the alfalfabe expressed as soon as possible after plant harvest. More preferablestill is that the alfalfa be expressed less than 1 hour after plantharvest.

For small batches of juice (up to approximately 15 liters), the alfalfais first macerated in any suitable type of mechanical macerator, such asan impact macerator. The juice is then immediately expressed using anytype of suitable means for pressing. For small batches, a hand-poweredhydraulic basket press or screw press is suitable. For larger volumes ofplant material, industrial-sized power equipment is required. Any fiberor debris is removed by filtration.

Replacement of Supplemental Phosphate with Juice from Transgenic AlfalfaExpressing Phytase Activity

As illustrated in the Examples below, the present inventors have shownthat when supplemental phosphate is removed from a basal chicken diet,the chicks quickly develop severe clinical signs of phosphorousdeficiency within the first week of life. Chicks which were fed a dietsupplemented with alfalfa juice from non-transformed alfalfa alsodeveloped severe clinical signs of phosphorous deficiency. However,chicks fed an identical basal diet supplemented with alfalfa juice fromtransformed alfalfa which expresses phytase activity thrived. Thisclearly illustrates that the added alfalfa juice, which contains phytaseactivity, increases the bio-availability of phosphorous present inphytate in the basal diet to the point that supplementation of theration with inorganic phosphorous is no longer necessary.

Feed Composition Containing Transgenic Alfalfa

In light of the above finding, one aspect of the present invention is amonogastric animal feed ration which includes dry plant matter or juicefrom transgenic alfalfa which expresses exogenous phytase. The feedration may contain dried transgenic alfalfa per se, whole juiceextracted from the transgenic alfalfa, frozen juice extracted from thetransgenic alfalfa, concentrated juice extracted from the transgenicalfalfa, or fractionated portions of the alfalfa juice. The alfalfa perse, or the alfalfa juice or concentrated juice can be added to standardmonogastric feed rations, such as the poultry ration described inExample 4 below. Preferably, the transgenic alfalfa or the juice of thetransgenic alfalfa is added in quantities sufficient to eliminate theneed to supplement the base ration with inorganic phosphorous.

The feed composition may also include leafy plant material or juice fromnon-transformed alfalfa as well. This is particularly applicable to fowlrations, which conventionally include xanthophyll supplementation topromote good skin and egg yolk coloration. The desired xanthophyllsupplementation (as well as additional dietary protein) can beincorporated into the ration by adding non-transformed alfalfa oralfalfa juice to the feed, or by adding xanthophyll extracted from juiceproducts where pigmenting is not desired.

Stability of the Phytase Enzyme From Transgenic Alfalfa

Fresh alfalfa juice from transgenic plants expressing phytase asdescribed above were tested for phytase activity after treatment at 0°C., 30° C. and 50° C. over a period of 100 minutes. In all cases,phytase activity within the alfalfa juice was not detectably diminishedafter 100 minutes of incubation at the stated temperature. (See FIG. 2.)As shown in FIG. 2, the empty squares represent the samples treated at0° C., the diamonds represent the samples treated at 30° C. and thesolid squares represent the samples treated at 50° C.

Additionally, juice extracted from transgenic alfalfa and thenconcentrated retains approximately 88% of its initial phytase activityover a period of at least one month when stored at room temperature(about 22° C.).

The remarkable stability of the recombinant phytase enzyme from alfalfamakes the industrial application of this invention very attractive.Additionally, the fibrous material remaining after juice extraction alsocontains residual phytase. This residual phytase may also have feedvalue when fed to ruminants.

EXAMPLES

In order to more fully illustrate the present invention, the followingExamples are provided. The Examples, which make reference to theattached Figures, are for illustration purposes only, to aid in a morecomplete understanding of the invention. The Examples do not limit thescope invention disclosed and claimed herein in any fashion.

Example 1

Expected yields

This Example reports the expected yields of various alfalfa juicefractions, including phytase and xanthophylls, isolatable fromtransgenic alfalfa which constituitively expresses exogenous phytaseoriginating from A. niger. Freshly expressed alfalfa juice was analyzedno less than 140 times on 40 days spanning a single growing season inDane County, Wis., extending from mid-June to mid-October. The resultsare presented in FIG. 1.

Each box of FIG. 1 includes two designations. The first designation isthe amount of the respective fraction based upon 1000 kg of harvesteddry matter. The second designation is the amount of the respectivefraction based upon an expected per acre annual yield. This designationis placed in parentheses (). This Example tabulates the completeisolation of soluble and particulate protein, phytase, and xanthophylls.Complete isolation of the individual components present in thetransgenic alfalfa, as illustrated here, is not necessary to exploit theadvantages of the transgenic alfalfa. This Example is solely toillustrate the relative amounts of soluble protein, particulate protein,phytase and xanthophylls which can be recovered from transgenic alfalfa.

As shown in boxes 2 and 3 of FIG. 1, each 1000 kg of dry matter (d.m.)after expression of the juice contained therein yields on average 700 kgof fibrous matter and 300 kg of juice, respectively. The fibrous mattercan be used for any number of purposes, including as a feed forruminants, a feedstock for biomass or ethanol production, a mulch, etc.

As depicted in box 4, the soluble protein fraction makes upapproximately 15 % by weight of the juice (dry matter basis), 90% ofwhich is, on average, protein. Particulate proteins make upapproximately 40% by weight of the juice, 45 % (average) of which isprotein (box 5), and 45% by weight of the juice dry matter is made up ofother soluble components such as salts, sugars and the like (box 6).

Referring now to box 7, approximately 40.5 kg of soluble protein can beisolated per 1000 kg of dry herbage. Box 9 tabulates the amount ofphytase which is contained within the soluble protein fraction as afunction of the percent phytase present in the soluble protein fraction.For instance, if the soluble protein fraction contains 0.01 weightpercent phytase, approximately 4.05 grams of phytase, on average, can beisolated from 1000 kg of dry herbage. If the soluble protein fractioncontains 1.00 weight percent phytase, approximately 405 grams ofphytase, on average, can be isolated from 1000 kg of dry herbage. Thehigher producing transformants described herein have had phytase yieldsgreater than 1 % of the soluble protein.

Boxes 9 and 10 detail the amount of particulate protein and xanthophyllswhich are found, on average, in alfalfa juice, respectively. As noted inbox 10, the amount of xanthophylls present is calculated as a functionof the total amount of soluble protein plus particulate proteinconcentrates isolated from the juice. The amount of xanthophyllsrecovered is directly proportional to the sum of soluble proteins plusparticulate proteins. Consequently, as the amount of total proteinincreases, so does the amount of xanthophylls recovered.

Example 2

Non-ruminant feed supplement

Using the above average yields, the following conclusions can be drawnregarding the ability to supplement non-ruminant animal feeds withtransgenic alfalfa which expresses exogenous phytase activity.

First, regarding phytase activity and the requirements of poultry andswine, the following requirements are generally accepted in the animalnutrition art:

1 unit phytase=1 μmole PO₄ released/min @ pH 5.5 and 37° C.

Pure phytase=100 to 140 units per mg dry matter

Poultry require a minimum of 300 to 500 units phytase per kg ration,which corresponds to approximately 3.3 mg phytase per kg of ration, or3.3 g phytase per metric ton of ration.

Swine require approximately 1000 to 1200 units phytase per kg ration,which corresponds to approximately 9.2 mg phytase per kg of ration, or9.2 g phytase per metric ton of ration.

For this Example, poultry were considered to require 29 mg xanthophyllper kg ration, or 29 g xanthophyll per metric ton ration. Swine do notrequire xanthophyll.

The ratio of xanthophyll to phytase in poultry rations should preferablyapproximate a ration of 8.6:1 xanthophyll to phytase. This ratio isapproximated when the xanthophyll yield is about 1,000 mg/kg of boxes 4and 5 of the Figure (i.e. the fraction of soluble vs. particulateproteins isolated from the juice), and phytase is produced at the rateof approximately 0.5 % of the soluble protein fraction (box 7 of FIG.1).

At these rates, one acre of transgenic alfalfa produces enough phytaseto supplement 30.4 metric tons of poultry feed. At a higher phytaseyield of 0.5% phytase in the soluble protein fraction, the per acreyield of phytase is sufficient to supplement 304 metric tons of poultryfeed.

Since swine rations require approximately 2.5 times the amount ofphytase as compared to poultry rations, one acre of transgenic alfalfayielding 0.5% phytase in the soluble protein fraction producessufficient phytase to supplement 120 metric tons of swine ration.

It should also be noted that since swine do not require xanthophylls,there is economic potential to remove the xanthophylls (by partialfractionation or any other known means) present in the multi-componentcomposition in the event that the composition is destined for swinefeed.

Example 3

Storage stability when formulated into poultry feed

Both lyophilized and frozen whole alfalfa juice were used to supplementa standard poultry diet such that phytase activity of the feed was 400units/Kg (1 unit=quantity of enzyme required to liberate 1 μmoleinorganic phosphorus per minute from an excess of sodium phytate at 37°C., pH 5.5). Portions of treated feed were stored at 22° C., 4° C., and-20° C. After 1, 2 and 3 weeks of storage at these temperatures,representative samples were taken from each feed portion and assayed forphytase activity. The results of this Example are depicted in FIG. 3. Asshown in FIG. 3, results for the feed portions supplemented with frozenalfalfa juice from transgenic alfalfa are designated "juice"; resultsfor the feed portions supplemented with lyophilized juice fromtransgenic alfalfa are designated "lyoph. juice". The temperature underwhich each sample was stored is designated along the X-axis, and theY-axis designates the phytase activity within each sample. Thedesignations 10/15, 10/22, and 10/30 correspond to week 1, week 2, andweek 3 measurements, respectively. No clear evidence of a loss inphytase activity over the 3 weeks of the experiment is evident.

The stability of transgenic phytase in poultry feed was also confirmedin the chicken feeding trials described below. During those trials, thephytase activity of feed samples removed and frozen on day 1 and day 11was measured. Determined activities were 256 units/kg and 248 units/kg,respectively. Although the measured activities were lower than theexpected 400 units/kg (possibly due to interference in the assay fromfeed constituents), no significant loss of activity was observed.

Example 4

Chicken feeding trials

In this Example, 6 populations of 25 chicks each were fed base dietswhich were either unsupplemented with inorganic phosphate, supplementedwith various levels of inorganic phosphate, supplemented with wholejuice from non-transformed alfalfa, or supplemented with whole juicefrom alfalfa transformed to express phytase as described above. Thefeeding trial continued for three weeks. At day 5 of the study, thepopulations fed unsupplemented rations or rations supplemented withnon-transformed alfalfa juice displayed severe malnutrition and wereeuthanized.

The studies used one-day old male broilers obtained from NorthernHatcheries, Beaver Dam, Wisconsin. The broilers were vaccinated at day 1against IBD and Mareks. The base diet from which all of the treatmentdiets were formulated was as follows:

    ______________________________________     Base Diet Formulation:    ______________________________________    Premix            g/3 kg    ______________________________________    Vitamin A (10,000 IU/g)                      135    Vitamin D.sub.3 (8900 IU/g)                      20    Vitamin E         27    Riboflavin (100 g/lb)                      7.3    Vitamin B.sub.12 (300 mg/lb)                      3.6    ZnSO.sub.4 (36%)  20    MnO               19    Niacin (50%)      22    Pantothenic acid (25%)                      28    Choline (60%)     287    Bring to 3000 g with ground corn.    ______________________________________    Base Diet      kg/105 kg    ______________________________________    Corn           51.19    Soybean Meal   42.43    CaCO.sub.3     1.47    Corn Oil       7.23    Methionine     0.24    Salt           0.53    Premix         1.05    ______________________________________

Treatment diets:

The treatment diets, numbered 1 through 6, were formulated using thebase diet as follows:

    ______________________________________    1. 0%† monocalcium phosphate                       570 g granite grit    2. 33%† monocal. phos.                       188 g monocal and 382 g grit    3. 66%† monocal phos.                       376 g monocal and 194 g grit    4. 100%† monocal phos.                       570 g monocal    5. Non-T* Juice    410 g non-T juice + 160 g grit    6. T** juice       410 g T juice + 160 g grit    ______________________________________     †percent of NRC recommended allowance for monocalcium phosphate     *1.17 wt % juice from nontransformed alfalfa     **1.17 wt % juice from transformed alfalfa which expresses phytase

Each treatment was added to 34.43 kg of feed from the base diet.

                  TABLE 1    ______________________________________    Weight Gain in Chicks Fed Basal Diets Supplemented with Inorganic    Phosphorous or Juice from Transgenic Alfalfa    Diet             % Total    % Added Added    Phos-   % Available                                     %     0-2 week    Mono-Cal-P            Juice    phorus  Phosphorus                                     Calcium                                           wt gain (g)    ______________________________________    1. 0    0        0.38    0.13    0.67  Died @ d 5    2. 0.53 0        0.49    0.24    0.76  243    3. 1.06 0        0.61    0.36    0.85  272    4. 1.60 0        0.72    0.47    0.94  302    5. 0    1.17%    0.38    0.13    0.67  Died @ d 5            Non-T*    6. 0    1.17%    0.38    0.13    0.67  244            trans-            formed**    ______________________________________     *1.17% juice from normal, nontransformed alfalfa     **1.17% juice from transgenic alfalfa expressing phytase activity

Utilizing this data, a mathematical regression of % monocalciumphosphate added versus weight gain reveals that in this Example R²=0.985 (weight gain in grams) and y=213.66+55.176 (% monocalciumphosphate). Therefore, based on equivalent weight gain, the juice fromtransgenic alfalfa increased the available phosphorous to the sameextent as adding 0.55% monocalcium phosphate to the ration.

Week by week average weights and average weight gains for the 6populations are reported in Table 2. Week by week feed-to-gain ratios,as well as the feed-to-gain ratios for the entire three-week study arepresented in Table 3.

Example 5

Estimated economic impact on chicken production in the United Statesreplacing supplemental phosphate with phytase derived from transgenicalfalfa

For this Example, the following estimates were employed:

6 billion broilers produced annually in the United States;

average market weight equals 4 lbs. per broiler;

1.85 feed conversion ratio (1.85 lbs of feed/lb weight gain);

average cost of supplemental phosphorous (monocalcium phosphate) equals$300/ton; and

replacement value of phytase=0.55% added monocalcium phosphate.

                                      TABLE 2    __________________________________________________________________________    Average weight gains       Avg. Wt.            Avg. Wt.                 0-1 Week                      Avg. Wt.                           0-2 Week                                1-2 Week                                     Avg. Wt.                                          0-3 Week                                               2-3 Week    Diet       Day 1            Week 1                 Wt. Gain                      Week 2                           Wt. Gain                                Wt. Gain                                     Week 3                                          Wt. Gain                                               Wt. Gain    __________________________________________________________________________    1  43   --   --   --   --   --   --   --   --    2  40   123  83   285  245  162  569  504  271    3  41   122  81   309  268  184  647  604  323    4  43   130  87   350  304  211  640  597  314    5  44   --   --   --   --   --   --   --   --    6  40   126  86   284  244  158  530  492  246    __________________________________________________________________________

                  TABLE 3    ______________________________________    Feed conversion ratios (feed-to-weight gain ratios)                                     2-3 Week                                            0-3 Week         Week 1    1-2 Week  0-2 Week                                     Feed/  Feed/    Diet Feed/Grain                   Feed/Grain                             Feed/Grain                                     Grain  Grain    ______________________________________    1    --        --        --      --     --    2    1.167     1.469     1.365   1.546  1.495    3    1.340     1.227     1.244   1.481  1.345    4    1.374     1.372     1.357   1.598  1.515    5    --        --        --      --     --    6    1.336     1.722     1.560   1.271  1.431    ______________________________________

Armed with the above assumptions, the annual economic value of phytaseproduced in transgenic alfalfa in the poultry industry in the UnitedStates is estimated as follows: ##EQU1##

This number does not take into account the value of the xanthophyll andthe dietary protein contained in the juice. In addition, the level ofphytase in the ration was assumed to be 250 U/kg rather than the target400 U/kg, suggesting that the juice might be used at almost double thelevel calculated above.

The estimated acreage required to produce 260,057 tons of alfalfa juice,the amount which would be needed in the above calculation, is 26,000acres, which equals approximately 1% of the State of Wisconsin's alfalfaacreage.

If the juice concentration is doubled for poultry rations and increased2.5 times for swine rations as compared to the poultry ration, theestimates calculted above would appear as follows:

    ______________________________________    Value of phytase alfalfa juice =                          $256 million    Amount of alfalfa juice required =                          1.82 million tons    Acreage required =    182,000    % of Wisconsin alfalfa acreage =                          6%    ______________________________________

Example 6

Pigment content of whole alfalfa juice

Frozen alfalfa juice from transgenic alfalfa (produced as describedabove) and non-transgenic alfalfa was analyzed for pigment content asdescribed by Thayer & Bjorkman (1990). Average values of xanthophyllwere 179 mg/kg whole juice (non-transformed control) and 123 mg/kg(transgenic alfalfa expressing phytase). These values correspond toapproximately 1500 mg/kg and 1025 mg/kg juice dry matter, respectively(based upon 12% dry matter in juice).

Based on these values, xanthophyll pigment content of poultry feedcontributed by non-transformed (control) alfalfa juice was 74 mg ofpigment per 35 kg of feed and xanthophyll content of poultry feedcontributed by transformed alfalfa juice was 50 mg pigment per 35 kg offeed.

Previous work by others has shown that a level of approximately 1 gramof xanthophyll per 35 kg of feed is optimal for skin pigmentation inbroilers. To achieve similar levels of xanthophyll using whole alfalfajuice would require adding approximately 800 grams of dried transgenicalfalfa juice per 35 kg of feed. Although this is more than theapproximately 500 grams of inorganic phosphate which is normally addedto the same quantity of feed, the alfalfa juice dry matter alsocontributes a significant quantity of protein. This indicates that thejuice would partly replace the need to add other exogenous sources ofxanthophyll to the ration.

Example 7

Stability of transgenic phytase in dried plant material

Experiments using transgenic tobacco expressing phytase showed thatapproximately 30% of the enzyme can be recovered from dried leafmaterial by simply grinding and rehydrating the leaves. The recoveredactivity has the same relative activity at high temperature and low pH,indicating that drying does not adversely affect the enzyme. Phytaseexpressed in alfalfa behaves in the same fashion.

It is understood that the invention is not confined to the particularmethodologies herein illustrated and described, but embraces suchmodified forms thereof as come within the scope of the following claims.

BIBLIOGRAPHY

Austin et al. (1994), An overview of a feasibility study for theproduction of industrial enzymes in transgenic alfalfa, Annals N.Y.Acad. of Sci., (721):234-242.

Austin et al. (1995), Euphytica, 00: 1-13.

Comai et al. (1990), Plant Mol. Biol. 15:373-381.

Ehrlich et al. (1993), Biochem. & Biophys. Res. Comm., 195(1):53-57.

Gibson, D. M. and Ullah, A. B. J. (1990), Inositol metabolism in plants,Wiley-LISS, Inc., 77-82.

Han et al. (1995), Proceedings of the 1995 Cornell Nutrition Conferencefor Feed Manufacturers, Rochester, New York, pp. 149-152.

Hughes, G. P. and Eyles, D. E. (1952), Extracted Herbage Leaf Proteinfor Poultry Feeding, J. Agric. Sci (Cambridge), 43:136-143.

Thayer & Bjorkman (1990), Photosynth. Res. 23:331-343.

Verwoerd et al. (1995), Plant Physiology, 109:1199-1205.

What is claimed is:
 1. An animal feed composition comprising juice,frozen juice, or concentrated juice of transgenic alfalfa plants whichconstituitively express exogenous phytase activity at a concentration ofat least about 0.5% percent by weight of soluble protein present in thetransgenic alfalfa.
 2. The animal feed composition of claim 1, whereinthe transgenic alfalfa expresses phytase at a concentration of fromabout 1.0% to about 2.0% by weight of soluble protein present in thetransgenic alfalfa.
 3. The animal feed composition of claim 1 which is apoultry feed composition, the poultry feed composition furthercomprising an inorganic phosphorous-free base ration suitable foradministration to poultry.
 4. The animal feed composition of claim 1which is a swine feed composition, the swine feed composition furthercomprising an inorganic phosphorous-free base ration suitable foradministration to swine.
 5. The animal feed composition of claim 1 whichis a ruminant feed composition, the ruminant feed composition furthercomprising an inorganic phosphorous-free base ration suitable foradministration to ruminants.
 6. The animal feed composition of claim 1which is a fish feed composition, the fish feed composition furthercomprising an inorganic phosphorous-free base ration suitable foradministration to fish.
 7. The animal feed composition of claim 1, whichcontains alfalfa juice from which xanthophylls have been removed.
 8. Theanimal feed composition of claim 1, further comprising juice, frozenjuice, or concentrated juice of non-transgenic alfalfa plants.
 9. Amethod of feeding livestock, poultry, or fish which reduces oreliminates the need for phosphorous supplementation of livestock,poultry, or fish rations comprising: feeding livestock, poultry, or fishan animal feed composition of claim
 1. 10. A method of feedingmonogastric livestock or poultry which reduces or eliminates the needfor phosphorous supplementation of monogastric livestock or poultryrations comprising: feeding monogastric livestock an animal feedcomposition of claim 1.